Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

Rapid Detection of SARS-CoV-2 RNA in Human Nasopharyngeal Specimens Using Surface-Enhanced Raman Spectroscopy and Deep Learning Algorithms.

A rapid and cost-effective method to detect the infection of SARS-CoV-2 is fundamental to mitigating the current COVID-19 pandemic. Herein, a surface-enhanced Raman spectroscopy (SERS) sensor with a deep learning algorithm has been developed for the rapid detection of SARS-CoV-2 RNA in human nasopharyngeal swab (HNS) specimens. The SERS sensor was prepared using a silver nanorod array (AgNR) substrate by assembling DNA probes to capture SARS-CoV-2 RNA. The SERS spectra of HNS specimens were collected after RNA hybridization, and the corresponding SERS peaks were identified. The RNA detection range was determined to be 103-109 copies/mL in saline sodium citrate buffer. A recurrent neural network (RNN)-based deep learning model was developed to classify 40 positive and 120 negative specimens with an overall accuracy of 98.9%. For the blind test of 72 specimens, the RNN model gave a 97.2% accuracy prediction for positive specimens and a 100% accuracy for negative specimens. All the detections were performed in 25 min. These results suggest that the DNA-functionalized AgNR array SERS sensor combined with a deep learning algorithm could serve as a potential rapid point-of-care COVID-19 diagnostic platform.

Nebulized mRNA-Encoded Antibodies Protect Hamsters from SARS-CoV-2 Infection.

Despite the success of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines, there remains a clear need for new classes of preventatives for respiratory viral infections due to vaccine hesitancy, lack of sterilizing immunity, and for at-risk patient populations, including the immunocompromised. While many neutralizing antibodies have been identified, and several approved, to treat COVID-19, systemic delivery, large doses, and high costs have the potential to limit their widespread use, especially in low- and middle-income countries. To use these antibodies more efficiently, an inhalable formulation is developed that allows for the expression of mRNA-encoded, membrane-anchored neutralizing antibodies in the lung to mitigate SARS-CoV-2 infections. First, the ability of mRNA-encoded, membrane-anchored, anti-SARS-CoV-2 antibodies to prevent infections in vitro is demonstrated. Next, it is demonstrated that nebulizer-based delivery of these mRNA-expressed neutralizing antibodies potently abrogates disease in the hamster model. Overall, these results support the use of nebulizer-based mRNA expression of neutralizing antibodies as a new paradigm for mitigating respiratory virus infections.

Rapid and quantitative detection of respiratory viruses using surface-enhanced Raman spectroscopy and machine learning.

Rapid and sensitive pathogen detection is important for prevention and control of disease. Here, we report a label-free diagnostic platform that combines surface-enhanced Raman scattering (SERS) and machine learning for the rapid and accurate detection of thirteen respiratory virus species including SARS-CoV-2, common human coronaviruses, influenza viruses, and others. Virus detection and measurement have been performed using highly sensitive SiO2 coated silver nanorod array substrates, allowing for detection and identification of their characteristic SERS peaks. Using appropriate spectral processing procedures and machine learning algorithms (MLAs) including support vector machine (SVM), k-nearest neighbor, and random forest, the virus species as well as strains and variants have been differentiated and classified and a differentiation accuracy of >99% has been obtained. Utilizing SVM-based regression, quantitative calibration curves have been constructed to accurately estimate the unknown virus concentrations in buffer and saliva. This study shows that using a combination of SERS, MLA, and regression, it is possible to classify and quantify the virus in saliva, which could aid medical diagnosis and therapeutic intervention.

Probenecid Inhibits Respiratory Syncytial Virus (RSV) Replication.

RNA viruses like SARS-CoV-2, influenza virus, and respiratory syncytial virus (RSV) are dependent on host genes for replication. We investigated if probenecid, an FDA-approved and safe urate-lowering drug that inhibits organic anion transporters (OATs) has prophylactic or therapeutic efficacy to inhibit RSV replication in three epithelial cell lines used in RSV studies, i.e., Vero E6 cells, HEp-2 cells, and in primary normal human bronchoepithelial (NHBE) cells, and in BALB/c mice. The studies showed that nanomolar concentrations of all probenecid regimens prevent RSV strain A and B replication in vitro and RSV strain A in vivo, representing a potential prophylactic and chemotherapeutic for RSV.

Exploring Noncovalent Protease Inhibitors for the Treatment of Severe Acute Respiratory Syndrome and Severe Acute Respiratory Syndrome-Like Coronaviruses.

Over the last 20 years, both severe acute respiratory syndrome coronavirus-1 and severe acute respiratory syndrome coronavirus-2 have transmitted from animal hosts to humans causing zoonotic outbreaks of severe disease. Both viruses originate from a group of betacoronaviruses known as subgroup 2b. The emergence of two dangerous human pathogens from this group along with previous studies illustrating the potential of other subgroup 2b members to transmit to humans has underscored the need for antiviral development against them. Coronaviruses modify the host innate immune response in part through the reversal of ubiquitination and ISGylation with their papain-like protease (PLpro). To identify unique or overarching subgroup 2b structural features or enzymatic biases, the PLpro from a subgroup 2b bat coronavirus, BtSCoV-Rf1.2004, was biochemically and structurally evaluated. This evaluation revealed that PLpros from subgroup 2b coronaviruses have narrow substrate specificity for K48 polyubiquitin and ISG15 originating from certain species. The PLpro of BtSCoV-Rf1.2004 was used as a tool alongside PLpro of CoV-1 and CoV-2 to design 30 novel noncovalent drug-like pan subgroup 2b PLpro inhibitors that included determining the effects of using previously unexplored core linkers within these compounds. Two crystal structures of BtSCoV-Rf1.2004 PLpro bound to these inhibitors aided in compound design as well as shared structural features among subgroup 2b proteases. Screening of these three subgroup 2b PLpros against this novel set of inhibitors along with cytotoxicity studies provide new directions for pan-coronavirus subgroup 2b antiviral development of PLpro inhibitors.

Silver nanotriangle array based LSPR sensor for rapid coronavirus detection.

A rapid, portable, and cost-effective method to detect the infection of SARS-CoV-2 is fundamental toward mitigating the current COVID-19 pandemic. Herein, a human angiotensin-converting enzyme 2 protein (ACE2) functionalized silver nanotriangle (AgNT) array localized surface plasmon resonance (LSPR) sensor is developed for rapid coronavirus detection, which is validated by SARS-CoV-2 spike RBD protein and CoV NL63 virus with high sensitivity and specificity. A linear shift of the LSPR wavelength versus the logarithm of the concentration of the spike RBD protein and CoV NL63 is observed. The limits of detection for the spike RBD protein, CoV NL63 in buffer and untreated saliva are determined to be 0.83 pM, 391 PFU/mL, and 625 PFU/mL, respectively, while the detection time is found to be less than 20 min. Thus, the AgNT array optical sensor could serve as a potential rapid point-of-care COVID-19 diagnostic platform.

Probenecid inhibits SARS-CoV-2 replication in vivo and in vitro.

Effective vaccines are slowing the COVID-19 pandemic, but SARS-CoV-2 will likely remain an issue in the future making it important to have therapeutics to treat patients. There are few options for treating patients with COVID-19. We show probenecid potently blocks SARS-CoV-2 replication in mammalian cells and virus replication in a hamster model. Furthermore, we demonstrate that plasma concentrations up to 50-fold higher than the protein binding adjusted IC90 value are achievable for 24 h following a single oral dose. These data support the potential clinical utility of probenecid to control SARS-CoV-2 infection in humans.

Selinexor, a novel selective inhibitor of nuclear export, reduces SARS-CoV-2 infection and protects the respiratory system in vivo.

The novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the recent global pandemic. The nuclear export protein (XPO1) has a direct role in the export of SARS-CoV proteins including ORF3b, ORF9b, and nucleocapsid. Inhibition of XPO1 induces anti-inflammatory, anti-viral, and antioxidant pathways. Selinexor is an FDA-approved XPO1 inhibitor. Through bioinformatics analysis, we predicted nuclear export sequences in the ACE-2 protein and confirmed by in vitro testing that inhibition of XPO1 with selinexor induces nuclear localization of ACE-2. Administration of selinexor inhibited viral infection prophylactically as well as therapeutically in vitro. In a ferret model of COVID-19, selinexor treatment reduced viral load in the lungs and protected against tissue damage in the nasal turbinates and lungs in vivo. Our studies demonstrated that selinexor downregulated the pro-inflammatory cytokines IL-1ß, IL-6, IL-10, IFN-γ, TNF-α, and GMCSF, commonly associated with the cytokine storm observed in COVID-19 patients. Our findings indicate that nuclear export is critical for SARS-CoV-2 infection and for COVID-19 pathology and suggest that inhibition of XPO1 by selinexor could be a viable anti-viral treatment option.

Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents.

Cas13a has been used to target RNA viruses in cell culture, but efficacy has not been demonstrated in animal models. In this study, we used messenger RNA (mRNA)-encoded Cas13a for mitigating influenza virus A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in mice and hamsters, respectively. We designed CRISPR RNAs (crRNAs) specific for PB1 and highly conserved regions of PB2 of influenza virus, and against the replicase and nucleocapsid genes of SARS-CoV-2, and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. We delivered polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract using a nebulizer. In mice, Cas13a degraded influenza RNA in lung tissue efficiently when delivered after infection, whereas in hamsters, Cas13a delivery reduced SARS-CoV-2 replication and reduced symptoms. Our findings suggest that Cas13a-mediated targeting of pathogenic viruses can mitigate respiratory infections.